Prosaposin PS18 reduces dopaminergic neurodegeneration in a 6-hydroxydopamine rat model of Parkinson’s disease

Saposin and its precursor prosaposin are endogenous proteins with neurotrophic and anti-apoptotic properties. Prosaposin or its analog prosaposin-derived 18-mer peptide (PS18) reduced neuronal damage in hippocampus and apoptosis in stroke brain. Its role in Parkinson’s disease (PD) has not been well characterized. This study aimed to examine the physiological role of PS18 in 6-hydroxydopamine (6-OHDA) cellular and animal models of PD. We found that PS18 significantly antagonized 6-OHDA -mediated dopaminergic neuronal loss and TUNEL in rat primary dopaminergic neuronal culture. In SH-SY5Y cells overexpressing the secreted ER calcium—monitoring proteins, we found that PS18 significantly reduced thapsigargin and 6-OHDA-mediated ER stress. The expression of prosaposin and the protective effect of PS18 were next examined in hemiparkinsonian rats. 6-OHDA was unilaterally administered to striatum. The expression of prosaposin was transiently upregulated in striatum on D3 (day 3) after lesioning and returned below the basal level on D29. The 6-OHDA-lesioned rats developed bradykinesia and an increase in methamphetamine-mediated rotation, which was antagonized by PS18. Brain tissues were collected for Western blot, immunohistochemistry, and qRTPCR analysis. Tyrosine hydroxylase immunoreactivity was significantly reduced while the expressions of PERK, ATF6, CHOP, and BiP were upregulated in the lesioned nigra; these responses were significantly antagonized by PS18. Taken together, our data support that PS18 is neuroprotective in cellular and animal models of PD. The mechanisms of protection may involve anti-ER stress.


Results
PS18 induced neuroprotection in primary dopaminergic neuronal culture. Ventral mesencephalon (VM) tissues were harvested from rat fetal brains for primary dopaminergic neuronal culture. 6-OHDA, PS18, and vehicle were added to the culture on DIV10 (day-in-vitro 10, Fig. 1A). As seen in the representing photomicrographs ( Fig. 1B), 6-OHDA reduced the density of TH ( +) cell body and fibers. TH immunoreactivity (TH-ir) was averaged from all culture wells (n = 7 in each group). 6-OHDA significantly reduced TH-ir, which was significantly antagonized by PS18 ( Fig. 1B and C).

Discussions
In this study, we reported that PS18 antagonized 6-OHDA -mediated dopaminergic degeneration in primary rat ventral mesencephalic neuronal culture. 6-OHDA significantly reduced TH immunoreactivity and increased TUNEL. These responses were antagonized by PS18. The protective effect of PS18 was further demonstrated in unilaterally 6-OHDA-lesioned rats. PS18 improved locomotor activity, reduced rotational behavior, upregulated  www.nature.com/scientificreports/ www.nature.com/scientificreports/ striatal DAT expression, and increased nigral TH immunoreactivity in the lesioned animals. The major finding of our study is that PS18 is neuroprotective in cellular and animal PD models. 6-OHDA has been commonly used to examine the pathogenesis and therapy for PD 20 . 6-OHDA reduced mitochondrial activity, activated caspase-9 and caspase-3 21 , TUNEL, and programmed cell death, indicating that 6-OHDA induced neuronal degeneration through apoptosis. In this study, we showed that prosaposin PS18 reduced 6-OHDA-mediated TUNEL and the loss of TH-ir in dopaminergic neuronal culture. Our data thus support that PS18 induced protection by inhibiting apoptosis in dopaminergic neurons.
We demonstrated that striatal 6-OHDA lesioning time-dependently regulated the expression of prosaposin in nigra and striatum. 6-OHDA transiently increased the expression of striatal prosaposin on D3. A delayed upregulation of prosaposin on D29 was found in nigra. As prosaposin is protective against 6-OHDA, the upregulation of prosaposin may indicate time-dependent endogenous neuroprotection was turned on after lesioning striatum and nigra. However, this endogenous protection was insufficient as prosaposin mRNA was down-regulated in striatum on D29. Exogenous prosaposin PS18 was needed to antagonize the dopaminergic degeneration and improve the prosaposin expression on D29.
Using immunohistochemistry, we demonstrated that 6-OHDA significantly reduced TH immunoreactivity and cell number in nigra. Both responses were significantly antagonized by PS18. The protective effect of PS18 in nigra was further supported by the Western blot analysis. We demonstrated that TH protein expression was significantly reduced in the lesioned nigra, and PS18 significantly antagonized 6-OHDA -mediated TH protein loss in nigra. Our data suggest that PS18 partially preserved TH activity in the nigra and PS18 is neuroprotective against dopaminergic neuronal loss in nigra. We also found that PS18 improved the expression of DAT mRNA in lesioned striatum on day 29. However, it did not significantly alter striatal TH protein expression on day 5. The discrepancy of dopaminergic marker expression in striatum may be attributed to the difference in protein translation or the time-dependent protection in striatum, which warrant future study. ER stress is a major cause of dopaminergic degeneration in PD 27 . 6-OHDA-induced degeneration through ER stress 28 . We demonstrated that 6-OHDA significantly upregulated the expression of ER stress markers (i.e., PERK, ATF6, CHOP, and BiP) in the lesioned nigra. 6-OHDA also significantly increased BiP protein production in nigra. These responses were antagonized by PS18. To further characterize the interaction of PS18 and ER stress, SY5Y cells expressing ER stress reporter gLuc-SERCaMP were used 11,12 . PS18 significantly reduced Tg and 6-OHDA -mediated gLuc-SERCaMP release. Furthermore, Tg or 6-OHDA-mediated TUNEL was significantly antagonized by PS18 in dopaminergic neuronal culture. Altogether, these data suggest that the protective effect of PS18 against 6-OHDA-mediated neurodegeneration involves anti-ER stress.
We, and others, previously reported that mesencephalic astrocyte-derived neurotrophic factor (MANF) is responsive to ER stress 29 and has been considered an ER stress response protein 30,31 . MANF protected against ER stress-induced cell death 30 . In addition, MANF reduced ER stress by facilitating the formation of cysteine bridges and protein folding in the ER 32 . As the N-terminal domain of MANF is structurally similar to a saposin, a common anti-ER stress pathway may be involved in MANF and prosaposin-mediated protection.
There are a few limitations in this study. TH cells were counted from 4 brain slices between bregma −5.16 to −5.76 mm per each animal. A more intensive unbiased stereology analysis is needed. Systemically applied prosaposin PS18 cannot cross the blood-brain barrier. In addition, large peptides are often quickly metabolized by protease in the periphery. To overcome these limitations, we applied PS18 intracerebroventricularly to www.nature.com/scientificreports/ 6-OHDA -lesioned rats. As i.c.v. is invasive and may not be clinically relevant, the effectiveness of PS18 through other delivery routes, such as intranasal or dioleoylphosphatidylserine liposome delivery system 5 , warrants further investigation. In conclusion, we demonstrated that prosaposin PS18 induced neuroprotection against dopaminergic neurotoxin 6-OHDA in primary dopaminergic neuronal culture. Exogenous PS18 improved locomotor function, reduced rotational behavior, and improved nigral TH expression in hemiparkinsonian rats. The mechanisms of protection involve anti-apoptosis and anti-ER stress. Our data support the beneficial effects of prosaposin PS18nin PD animals. Further clinical studies are needed before clinical use.

Primary cultures of rat ventral mesencephalon cells.
Primary cultures were prepared from embryonic (E15) ventral mesencephalon (VM) tissues obtained from fetuses of timed-pregnant Sprague-Dawley rats. The whole brain was removed aseptically, and a small piece of tissue comprising the VM was dissected. After removing the blood vessels and meninges, pooled VM tissues were trypsinized (0.25%; Invitrogen, Carlsbad, CA) with gentle mixing for 15 min at 37 °C. After rinsing off trypsin with pre-warmed DMEM/F-12 (Invitrogen), cells were dissociated by trituration, counted, and plated into 96-well (6.0 × 10 4 /well) cell culture plates pre-coated with poly-D-lysine (Sigma-Aldrich). The culture plating medium consisted of Dulbecco's modified Eagle medium/F12 supplemented with 10% heat-inactivated fetal bovine serum, 1 mM L-glutamine, and 2% B27 (Invitrogen). Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cultures were fed by exchanging 50% of media with feed media (Neurobasal medium, Invitrogen) with 0.5 mM l-glutamate and 2% B27 with antioxidants supplement on DIV (days in vitro) 3 and 5.
Immunocytochemistry and quantitation. After removing the PFA solution, cells were washed with PBS, and the fixed cultures were treated for 1 h with blocking solution (2% BSA, 0. 1% Triton X-100 and 5% goat serum in PBS). The cells were incubated for 1 day at 4 °C with specific mono/polyclonal antibodies (i.e., TH) and then rinsed three times with PBS. The bound primary antibody was visualized using Alexa Fluor 488 secondary (Invitrogen). Images were acquired using a monochrome camera Qi1-mc attached to Nikon TE2000-E inverted microscope.
Gaussia luciferase secretion assay. Five microliters of culture medium were transferred to white 96-well plates. Coelenterazine (Cat# 1-361204-200Regis Technologies) stock solutions were prepared at 20 mM in acidified methanol (10ul of 10 N HCl/1 ml of methanol) and stored at -80 °C as single-use aliquots. The prepared substrate was incubated at room temperature 30 min prior to use. One hundred microliters of the diluted substrate were injected into each well followed by immediate luminescence reading. The amount of luciferase was determined using a plate reader with an injector setup (Biotek Synergy HT, Winooski, VT) to immediately read the sample after injection. For secretion assays, vehicle controls were used in all experiments under conditions equivalent to the treatments.

Terminal deoxynucleotidyl transferase (TdT)-mediated dNTP nick-end labeling (TUNEL). Cul-
tures were examined for DNA fragmentation using a TUNEL-based method (In Situ Cell Death Detection Kit; Roche, Indianapolis, IN). Briefly, 4% PFA fixed cells were permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. To label damaged nuclei, 50 μL of the TUNEL reaction mixture was added to each sample and kept at 37 °C in a humidified chamber for 60 min. Procedures for positive and negative controls were carried out as described in the manufacturer's manual (Roche). Controls consisted of not adding the label solution (terminal deoxynucleotidyl transferase) to the TUNEL reaction mixture. Nikon TE2000 inverted microscope equipped with fluorescence was used to examine apoptosis. www.nature.com/scientificreports/ Drug administration. Animals were anesthetized with 3% isoflurane. PS18 (2 mM/20 μL) or vehicle (saline, 20 μl) was administered intracerebroventricularly (AP, − 0.8 mm; LV, − 1.5 mm; DV, − 3.5 mm) at 15 min before 6-OHDA lesioning through a Hamilton microsyringe. The injection speed was controlled by a syringe pump (Micro 4, WPI, Sarasota, FL).

Behavioral test.
(1) Locomotor activity was examined on day 14 after 6OHDA lesioning. Rats were individually placed in 42 × 42 × 31 cm Plexiglas activity chambers containing horizontal and vertical infrared sensors (Accuscan, Columbus, OH) placed 2.5 cm apart. Three variables were measured: (i) horizontal activity (HACTV, the total number of beam interruptions that occurred in the horizontal sensors in one hour), (ii) vertical activity (VACTV, the total number of beam interruptions that occurred in the vertical sensor in one hour), and (iii) total distance traveled (TOTDIST, the distance, in centimeters, traveled by the animals in one hour). (2) Rotational behavior was evaluated using an 8-channel rotometer system (RotoMax, AccuScan Instruments, Inc). Meth (2.5 mg/kg)-induced rotation per hour was counted by a computer, as we previously described 33 .
Quantitative reverse transcription-PCR. Nigra   . Control sections were incubated without the primary antibody. Brain sections were mounted on slides and coverslipped. Confocal analysis was performed using a Nikon D-ECLIPSE 80i microscope (Nikon Instruments, Inc., Tokyo, Japan) and EZ-C1 3.90 software (Nikon, Tokyo, Japan). TH density (TH pixel density in striatum or nigra-the background density in cortex) was measured in brain sections with a visualized anterior commissure (for striatal TH) and in sections between −5.16 and −5.76 mm (for nigra). TH optical density was analyzed by NIS Elements AR 3.2 Software (Nikon) and was averaged in each brain for statistical analysis. TH( +) cell numbers in nigra were also examined in 4 brain sections between bregma −5.16 mm to −5.76 mm using NIS Elements AR 3.2 Software (Nikon). Only TH neurons with presence of nuclei were counted. All immunohistochemical measurements were performed by blinded observers.
Western blotting. The brain tissues were homogenized in RIPA lysis buffer (Merck Millipore, MA, USA) and then centrifuged at 13,200 rpm for 10 min at 4 °C. The supernatant was collected. A bicinchoninic acid (BCA) protein assay was performed to determine protein concentrations. The samples were diluted according to the BCA protein assay. Gels were transferred to a PVDF membrane after electrophoresis. The membranes were blocked in 5% milk at room temp for 1 h. The blots were then probed with primary antibodies against tyrosine hydroxylase (polyclonal, TH, 1: 10,000, Millipore, MA, USA), BiP (polyclonal, 1:1000, Cell Signaling, MA, USA), and actin (monoclonal, 1: 10,000, Novus, CO, USA) at 4 °C for overnight. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson lab) at room temp for 1 h, followed by washing with 0.1% Tween-20 (in PBS) three times for 10 min each. The light emission signal of the target proteins on the PVDF membrane was generated using a Western Lightning Plus-ECL (PerkinElmer, MA, USA) and then detected by X-ray film (Cat. No. GE28-9068-39, GE, Boston, USA). The amount of TH was normalized with actin on the same membrane. Band intensity was quantified using Image J.
Statistical analysis. Data are presented as the mean ± SEM. Unpaired t-test, one-or two-way ANOVA, and post-hoc Newman-Keuls (NK) test were used for statistical comparisons, with a significance level of p < 0.05. www.nature.com/scientificreports/ Ethical approval. All procedures and animal experiments were fully complied with the Animal Research Committee of the National Health Research Institutes of Taiwan (NHRI-IACUC-109097-M1). All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory Animals (NIH Publications No. 8023, revised 1978). This study is reported in accordance with ARRIVE guidelines.

Data availability
The datasets generated or analyzed in the current study are available from the corresponding authors on reasonable request (contact: b7508@nhri.edu.tw).